The present invention relates to veto cell preparations, methods of their manufacture and transplantation method using same which can be used to prevent or ameliorate immune rejection of donor organs, tissues or cells without inducing graft versus host disease (GVHD). More particularly, the present invention relates to a cell preparation for use in transplantation which preparation includes cells which are functional as veto cells but which are substantially devoid of alloreactive cells.
Transplantation of allogeneic and xenogeneic organs, tissues and cells is commonly practiced in humans in order to alleviate numerous disorders and diseases.
For example, bone marrow (BM) transplantation is increasingly used to treat a series of severe diseases in humans, such as for example, leukemia. However, bone marrow transplantation is limited by the availability of suitable donors, since transplanted tissues must traverse major histocompatibility barriers which can otherwise lead to graft rejection.
In view of such limitations, several approaches for enhancing graft acceptance have been suggested.
In one approach, cancer patients receiving autologous BM transplantation were treated with granulocyte colony-stimulating factor (G-CSF), resulting in mobilization of pluripotential stem cells from the marrow to the blood thereby increasing the number of cells which can be collected for autologous transplantation.
In another approach, major histocompatibility barriers in BM transplantation in leukemia patients were overcome by using a very large dose of stem cells, preferably a dose at least 3-fold greater than conventional doses used in T-cell-depleted BM transplantation, in particular a megadose of CD34+ hematopoietic progenitors (U.S. Pat. No. 5,806,529 to Reisner et al.).
Although the megadose approach facilitated permanent acceptance of allogeneic donor type skin grafts in mice 1, such an approach is not readily applicable for human transplantation since the number of stem cells which are required to attain this desirable goal may not be easily collected from human donors.
A difficult barrier for the engraftment of donor hematopoietic cell transplantation arises from the marked level of host hematopoietic and immune cells surviving mild preparatory regimens. Several studies have shown that this challenge can be successfully addressed in rodents by using large doses of bone marrow cells, adequately depleted of T-cells and utilized in conjunction with one form or another of tolerance inducing cells, also termed as veto cells.
Veto cell activity is defined as the capacity to specifically suppress cytotoxic T-cell precursors (CTL-p) directed against antigens of the veto cells themselves, but not against third party antigens 2 Several veto cells or bone marrow transplantation facilitating cells capable of suppressing cytotoxic T-cell precursors have been described 3-11.
Interestingly, it has been shown that some of the most potent veto cells are of T-cell origin, and in particular a very strong veto activity was documented for CD8+ CTL lines or clones 12-16.
The specificity of CTL veto cells was demonstrated by several studies to be unrelated to their T-cell receptor specificity 17-19.
The suppression of effector CTL-p directed against the veto cells is both antigen-specific and MHC-restricted. This suppression results from the unidirectional recognition of the veto cell by the responding cytotoxic T-lymphocytes 18. Furthermore, it has been shown that this suppression is mediated by apoptosis 18, 20.
Blocking experiments conducted with anti-CD8 or anti class I antibodies indicated that the elimination of host anti-donor CTL-p is induced via an interaction of CD8 molecules on the CTL veto cells with the a3 domain of class I molecules on the host CTL-p""s 18. Support to this observation was provided by studies in which CD8 cDNA was introduced into clones lacking CD8 18. Further support was provided by experiments which demonstrated that CD8 molecules on the veto cells can directly induce apoptosis in effector cells 21. More recently, Asiedu et al. demonstrated that antibody mediated cross linking of CD8 on primate bone marrow veto cells, leads to an increased TGFxcex2 production which induces apoptosis in the effector cells 22. Alternatively, it has been suggested that mouse bone marrow veto cells can induce apoptosis via Fas-Fas-L interaction 23.
The studies described hereinabove demonstrated that veto T-cell preparations can greatly facilitate graft tolerance in bone marrow transplantation. However, the veto T-cell preparations are inadequate for generating graft tolerance since such preparations still include a substantial amount of alloreacting donor T-cells which can lead to GVHD, thus limiting the successful implementation of this approach.
Reisner et al., 24 describe the preparation of non-alloreactive anti-third party CTLs which can be used to enhance graft acceptance in mice. However, following the publication of this abstract and a more careful study, it was realized that the CTL preparation described therein is not depleted of T-cells capable of developing post transplantation into anti-host CTLs inflicting GVHD. Thus, this approach per se is not applicable for application in humans.
There is thus a widely recognized need for, and it would be highly advantageous to have, a novel veto cell preparation devoid of alloreactivity which can be used for substantially reducing rejection of transplanted organs, tissues or cells without generating GVHD, thereby leading to durable tolerance towards the transplanted, organs tissues or cells.
According to one aspect of the present invention there is provided a method of transplanting a transplant derived from a donor into a recipient, the method comprising the steps of (a) transplanting the transplant into the recipient; and (b) administering to the recipient a dose including non-alloreactive anti-third party cytotoxic T-lymphocytes (CTLs), wherein the non-alloreactive anti-third party CTLs are generated by directing T-lymphocytes of the donor against a third party antigen or antigens, the dose is substantially depleted of T-lymphocytes capable of developing into alloreactive CTLs, thereby preventing or ameliorating both graft rejection by the recipient and graft versus host disease.
According to another aspect of the present invention there is provided a method of treating a recipient suffering from a disease requiring immature hematopoietic cell transplantation, the method comprising the steps of (a) conditioning the recipient under sublethal, lethal or supralethal conditions; (b) administering to the recipient a first dose including immature hematopoietic cells including stem cells from an allogeneic or xenogeneic donor; and (c) administering to the recipient a second dose including non-alloreactive anti-third party cytotoxic T-lymphocytes (CTLs), wherein the CTLs are generated by directing T-lymphocytes derived from the donor against a third party antigen or antigens, the second dose is substantially depleted of T-lymphocytes capable of developing into alloreactive CTLs, thereby preventing or ameliorating both graft rejection and graft versus host disease.
According to yet another aspect of the present invention there is provided a method of producing non-alloreactive anti-third party cytotoxic T-lymphocytes (CTLs), the method comprising the step of directing T-lymphocytes against a third party antigen or antigens, and substantially depleting T-lymphocytes capable of developing into alloreactive CTLs.
According to still another aspect of the present invention there is provided a cell preparation for transplantation to a recipient, the cell preparation comprising donor derived non-alloreactive anti-third party cytotoxic T-lymphocytes (CTLs) directed against a third party antigen or antigens, the cell preparation being substantially depleted of T-lymphocytes capable of developing into alloreactive CTLs.
According to an additional aspect of the present invention there is provided a cell preparation for transplantation to a recipient, the cell preparation comprising (a) donor derived immature hematopoietic cells including stem cells; and (b) donor derived, non-alloreactive anti-third party cytotoxic T-lymphocytes (CTLs) directed against a third party antigen or antigens, the cell preparation being substantially depleted of T-lymphocytes capable of developing into alloreactive CTLs.
According to yet an additional aspect of the present invention there is provided a method of transplanting a transplant derived from a donor into a recipient, the method comprising the steps of (a) transplanting the transplant into the recipient; and (b) administering to the recipient a dose including donor derived, genetically modified non-T-cells expressing recombinant Fas ligand and recombinant CD8 antigen, thereby serving as veto cells inherently lacking graft versus host activity and preventing or ameliorating graft rejection.
According to still an additional aspect of the present invention there is provided a method of treating a recipient suffering from a disease requiring immature hematopoietic cell transplantation, the method comprising the steps of (a) conditioning the recipient under sublethal, lethal or supralethal conditions; (b) administering to the recipient a first dose including immature hematopoietic cells including stem cells from an allogeneic or xenogeneic donor; and (b) administering to the recipient a second dose including donor derived, genetically modified non-T-cells expressing recombinant Fas ligand and recombinant CD8 antigen, thereby serving as veto cells inherently lacking graft versus host activity and preventing or ameliorating graft rejection.
According to a further aspect of the present invention there is provided a cell preparation for transplantation to a recipient, the cell preparation comprising donor derived, genetically modified non-T-cells expressing recombinant Fas ligand and recombinant CD8 antigen, thereby serving as veto cells inherently lacking graft versus host activity and preventing or ameliorating graft rejection.
According to yet a further aspect of the present invention there is provided a cell preparation for transplantation to a recipient, the cell preparation comprising (a) donor derived immature hematopoietic cells including stem cells; and (b) donor derived, genetically modified non-T-cells expressing recombinant Fas ligand and recombinant CD8 antigen, thereby serving as veto cells inherently lacking graft versus host activity and preventing or ameliorating graft rejection.
According to still further features in the described preferred embodiments depletion of T-lymphocytes capable of developing into alloreactive CTLs is effected by deprivation of a factor which is (i) required for CTLs maturation; and (ii) secreted by maturing CTLs.
According to still further features in the described preferred embodiments the factor is a cytokine.
According to still further features in the described preferred embodiments the cytokine is IL2.
According to still further features in the described preferred embodiments depletion of T-lymphocytes capable of developing into alloreactive CTLs is effected by affinity labeling followed by label based separation.
According to still further features in the described preferred embodiments depletion of T-lymphocytes capable of developing into alloreactive CTLs is effected by affinity purification.
According to still further features in the described preferred embodiments the donor is selected from the group consisting of an allogeneic donor either HLA identical or HLA non-identical and a xenogeneic donor.
According to still further features in the described preferred embodiments the recipient is a human.
According to still further features in the described preferred embodiments the recipient and the donor are both humans.
According to still further features in the described preferred embodiments the third party antigen or antigens is selected from the group consisting of third party cells, a cell antigen, a viral antigen, a bacterial antigen, a protein extract and a purified protein.
According to still further features in the described preferred embodiments the viral antigen is an EBV or a CMV antigen.
According to still further features in the described preferred embodiments the purified protein is ovalbumin.
According to still further features in the described preferred embodiments the third party cells are allogeneic or xenogeneic cells with respect to the recipient.
According to still further features in the described preferred embodiments the allogeneic cells have HLA antigens different from that of the donor but which are not cross reactive with the recipient HLA antigens.
According to still further features in the described preferred embodiments the allogeneic cells are stimulatory cells selected from the group consisting of cells purified from peripheral blood lymphocytes (PBLs), spleen or lymph nodes, cytokine-mobilized PBLs and in vitro expanded antigen-presenting dendritic cells (APC).
According to still further features in the described preferred embodiments the immature hematopoietic cells including stem cells are derived from the bone marrow, mobilized peripheral blood, fetal liver, yolk sac and/or cord blood of the donor.
According to still further features in the described preferred embodiments the mobilized peripheral blood cells are obtained by leukapheresis of peripheral blood of the donor after stimulation with a suitable cytokine.
According to still further features in the described preferred embodiments the immature hematopoietic cells are T-cell depleted hematopoietic progenitor cells.
According to still further features in the described preferred embodiments the T-cell depleted hematopoietic progenitor cells are CD34+ progenitor hematopoietic cells.
According to still further features in the described preferred embodiments a cell ratio between the cytotoxic T-lymphocytes and the immature hematopoietic cells including stem cells is at least 1 to 100, preferably 1.5 to 100.
According to still further features in the described preferred embodiments steps of transplanting and administering are effected at the same time, or alternatively, the step of transplanting is performed either prior to, or after the step of administering.
The present invention successfully addresses the shortcomings of the presently known configurations by providing veto cells which are highly effective in preventing graft rejection, yet are devoid of graft versus host disease potential.